History of West Mediterranean Newts, Pleurodeles (Amphibia
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Systematics and Biodiversity 1 (3): 327–337 Issued 16 February 2004 DOI: 10.1017/S1477200003001221 Printed in the United Kingdom C The Natural History Museum S. Carranza & E. N. Arnold∗ History of West Mediterranean newts, Department of Zoology, The Natural History Museum, Pleurodeles (Amphibia: Salamandridae), London, Cromwell Road, London, SW7 5BD, UK inferred from old and recent DNA submitted April 2003 accepted July 2003 sequences Abstract MtDNA sequences (396 bp cytochrome b and 369 bp 12S rRNA) from recent material and old museum specimens indicate Pleurodeles poireti and P. waltl form independent clades with 7.76% genetic divergence. Within P. poireti, populations from Djebel Edough, NE Algeria are very distinct with 6.12% genetic divergence from the remainder and may deserve separate species status. Away from Djebel Edough, P. poireti consists of three distinct clades (coastal NW Tunisia; central N Algeria; Constantine plus inland NW Tunisia) with a maximum genetic divergence of only 1%. P. waltl contains two clades with 2.96% genetic divergence, one in SE and E Spain plus north Morocco, the other in Portugal and SW and central Spain. Pleur- odeles probably invaded NW Africa from SW Europe during the Messinian Salinity Crisis, when land contact was first established at 5.6 Ma, and then interrupted at 5.3 Ma. Molecular clocks, calibrated in the assumption that the latter event separated P. waltl and P. poireti, suggest that Pleurodeles diverged from its sister taxon, Tylototriton, at about 8.6–10 Ma. Djebel Edough P. poireti separated at about 4.2 Ma, perhaps through isolation on a temporary, now ‘fossil’, island initiated by the Messinian crisis. Differentiation in remaining P. poireti may have been caused by Pleistocene climatic fluctuations, while bifurcation in P. waltl appears to have taken place in the Pliocene approximately between 3.2 and 2 Ma. This species reached Morocco very recently, perhaps as a result of human introduction. Use in Pleurode- les of the slower divergence rates estimated in some other salamandrids results in a less parsimonious historical hypothesis that does not fit known geophysical events. Key words ancient mtDNA, Pleurodeles, Tylototriton, Chelotriton,dispersal,land bridge, Messinian, fossil island, Djebel Edough Introduction often traces of this structure on ribs 1 and 2), and sharp rib tips that project through a row of glandular swellings on the In this paper we review the available fossil and other evidence flanks. P. waltl is common and widely distributed in the south- for the relationships and minimum age of the West Mediter- ern two-thirds of the Iberian Peninsula and occurs with less ranean salamandrid newt genus Pleurodeles, and use mtDNA abundance in northern Morocco, where it is found in the area sequence to investigate the detailed phylogeny of the group. delineated by Tangier, Casablanca and Alhoceima (Bons & The two sources of information are then employed to speculate Geniez, 1996). The second Pleurodeles species, P. poireti,is about the history of Pleurodeles including the origin of its small (up to about 200 mm in total length; Pasteur, 1958), with present biogeographical pattern. 14 presacral vertebrae, no obvious tubercular processes on the ribs which are not sharp, and no glandular swellings on the Current taxonomy of Pleurodeles flanks. It occurs in northern Tunisia and Algeria extending This genus has two currently recognized species. P. waltl is westwards as far as Oran. large (up to 300 mm in total length; Pasteur, 1958), with usu- ally 15 presacral vertebrae, a tubercular process on rib 3 (and Relationships and possible age of Pleurodeles Morphology and mitochondrial DNA sequences (a total of about 1000 bp of 12S rRNA, 16S rRNA and valine transfer *Corresponding author. Email: [email protected] RNA) indicate that among living forms, the sister taxon of 327 328 S. Carranza & E. N. Arnold Pleurodeles is the species of Tylototriton in South-East Asia, waltl from the Iberian Peninsula and Morocco and 23 P.poireti including the type species of that genus, T. verrucosus (Titus & from Algeria and Tunisia, while individuals of Salamandra sa- Larson, 1995). Within the Salamandridae, Pleurodeles and lamandra morenica, S. s. terrestris, Tylototriton taliangensis Tylototriton form an assemblage with a number of European and Mertensiella luschani were used as outgroups. Sequences fossil taxa (Group II of Estes, 1981), which is characterized of the last two species were obtained from Genbank. Ten of the by extensive sculptured dermal bone on the skull and some- P. poireti investigated were acquired between 1878 and 1888 times on the neural spines of the presacral vertebrae, complete by Ferdinand Lataste who bequeathed them, with the rest of frontosquamosal arches on the skull, and long ribs, at least his large herpetological collection, to George A. Boulenger at some of which typically have dorsal tubercular processes. One the Natural History Museum, London, where they were acces- genus in particular, the extinct Chelotriton, is very similar to sioned in 1920. Lataste apparently preserved his specimens in Tylototriton and was widespread in Europe from the Eocene a distilled wine product, probably brandy. To prevent evapor- to the Pliocene (Estes, 1981; Bailon, 1989). Both these taxa ation, he stored them in containers closed with a glass disc have extensive dermal bone on the skull and forming expanded attached with putty; this was then covered with parchment plaques on the neural spines of the vertebrae. Living Tyloto- that was tied with twine and finally sealed with black paint. In triton differ from Chelotriton in their smaller body size, an some cases, Lataste’s containers have been left unopened to the ornamentation of pits and grooves on the cranial osteoderms present day, so the enclosed specimens have remained in what instead of pustules (though some pitting may also occur in is essentially ethanol instead of being transferred to industrial Chelotriton robustus; Estes, 1981), typically 15–16 presacral methylated spirits (IMS), the usual preservative at the Natural vertebrae instead of 13–14, a tubercular process present only History Museum. Because of this there was the possibility on rib 3, ossified carpus and tarsus in at least large animals, that Lataste’s sealed specimens might still be viable sources of and a narrower head. DNA, which is usually largely or wholly degraded by IMS. To Tylototriton has been reported from the middle Eocene avoid contamination, Lataste’s animals were treated as ancient of Germany, as T. weigelti Herre 1935. Individuals assigned to DNA samples. The opening of the bottles, handling of speci- this taxon appear to have dermal ossification similar to that of mens, DNA extraction and PCR amplification were all carried modern Tylototriton, but the type is very small and all available out in isolation, following procedures previously successfully specimens are incomplete. This means several critical features used in other attempts to obtain DNA from old Museum speci- cannot be checked, including presacral number and degree mens (Carranza et al., 1999, 2001; Carranza & Arnold, 2003). of ossification of the carpus and tarsus. T. weigelti may in Nine of the ten Lataste specimens investigated yielded ad- fact be a Chelotriton (Milner, 2000). The earliest specimens equate DNA that was not seriously degraded, even though assigned to Pleurodeles are quite recent, coming from the they were collected well over a century ago. DNA was ex- Upper Miocene or Lower Pliocene of Spain (Sanchiz, 1977). tracted from both Lataste and recent samples using the Tissue It has been suggested (Herre, 1941) that the genus is related to Extraction Kit from Quiagene. Primers used in both ampli- Palaeopleurodeles Herre 1941 from the Upper Oligocene of fication and sequencing were: S1F (5- TTC AAC TAC AAA Germany, but several critical features cannot be seen in known AACCTAATGACCC-3) and cytochrome b2 (Kocher specimens of Palaeopleurodeles, so this association cannot be et al., 1989) for cytochrome b, and 12Sa and 12Sb (Kocher substantiated (see Estes, 1981; Milner, 2000). et al., 1989) for 12S rRNA. The molecular evidence for the sister relationship of Pleurodeles and Tylototriton is also supported by shared mor- Phylogenetic analyses phological features that are apparently derived within the Two data sets were used in the phylogenetic analyses. Data set Group II salamandrids, including an ossified carpus and tarsus, I involved all 42 specimens of Pleurodeles from Table 1, plus and sometimes 15 presacral vertebrae. The sister relationship Mertensiella luschani from Genbank and included 396 bp of indicates that the exclusive Pleurodeles lineage too may pos- the cytb and 369 bp of the 12S rRNA genes. Data set II involved sibly have arisen in the Upper Miocene. Its autapomorph- a representative of each of the six main Pleurodeles clades that ies include reduction of osteodermal bone (completely so on were discovered by analysis of data set I (highlighted in Figs 1 the vertebrae) and in the size of the tubercular processes and 2), plus S. s. terrestris, S. s. morenica, M. luschani and on the ribs. Comparison with Tylototriton and other sala- Tylototriton taliangensis; it included 346 bp of the cytb gene. mandrids indicates that the very large size and sharp ribs Analysis of this second data set was necessary to permit the of P. waltl are derived, as are the following features of P. inclusion of Tylototriton, the sister of Pleurodeles, because the poireti: small size, 14 presacral vertebrae, and lack of tuber- areas of sequence available for this taxon are different from cular processes and of glandular swellings on the side of the the other samples. Analysis of data set II provided a check that body. tree topology of Pleurodeles was not changed by inclusion of its closest relative and permitted an estimate to be made of the age of the node at which Pleurodeles and Tylototriton Material and Methods separated.